Toxoplasma gondii is an intracellular parasite that has infected up to one-third of the population. The pathogen can cause congenital birth defects and life-threatening opportunistic infection in HIV/AIDS. The replicative stage (tachyzoite) develops into a latent stage (bradyzoite) that resides inside brain, heart, and skeletal muscle tissues, and is impervious to immunity and currently approved antiparasitic drugs. Tissue cysts give rise to recurrent reactivation of infection in immunocompromised patients, creating chronic disease in HIV/AIDS patients. Despite its clinical importance, we know very little about the molecular details orchestrating the conversion between tachyzoites and bradyzoites. Studies have revealed that the development of bradyzoites, which can be triggered in vitro by cellular stress, is a complex process subject to regulation at transcriptional and post-transcriptional levels. Recently, it has been shown in other species that mRNA modifications, specifically post-transcriptional methylation of adenosines at position 6 (m6A), influence gene expression by altering RNA processing or translation. Changes in m6A marks have been associated with modulating cellular stress, development, and differentiation. The discovery of m6A represents a new layer of gene regulation called epitranscriptomics that has not been investigated in protozoan parasites. In preliminary studies, we establish that Toxoplasma RNA is subject to m6A, and we have begun characterizing the enzyme complex that delivers this RNA modification. The study of m6A in Toxoplasma in the context of HIV/AIDS is significant as this modification has been linked to stress-induced changes in cells, making it highly likely that m6A participates in bradyzoite development. In this new R21, we will address our hypothesis that m6A mRNA modifications are required for tachyzoite differentiation into bradyzoites by identifying changes in the m6A epitranscriptome during stage conversion (Aim 1) and determining the ?writer? protein complex that delivers m6A onto Toxoplasma mRNA (Aim 2). Our proposed studies will be the first analysis of m6A on mRNA in parasites, and will generate valuable datasets and reagents needed to interrogate this vital new area of investigation relevant to the development of the tissue cysts, which give rise to chronic toxoplasmosis in HIV/AIDS patients.